Methods and materials for multiplexed collections of functional ligands

ABSTRACT

This invention relates to methods and materials for multiplexed utilization of collections of functional ligands, particularly to methods and materials for selecting for and/or utilizing particular desirable traits of functional ligands in a multiplexed manner, and more particularly to methods and materials for selecting for and/or utilizing particular structural changes of functional ligands in a multiplexed manner.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit and priority of U.S. provisionalpatent application Ser. No. 62/624,063, filed Jan. 30, 2018, entitled“METHODS AND MATERIALS FOR MULTIPLEXED COLLECTIONS OF FUNCTIONALLIGANDS”, the contents of which is hereby incorporated by reference inits entirety.

COPYRIGHT NOTICE

A portion of the disclosure of this patent document contains materialwhich is subject to copyright protection. The copyright owner has noobjection to the facsimile reproduction by anyone of the patent documentor the patent disclosure, as it appears in the U.S. Patent and TrademarkOffice patent file or records, but otherwise reserves all copyrightrights whatsoever.

FIELD OF THE INVENTION

This invention relates to methods and materials for multiplexedutilization of collections of functional ligands, particularly tomethods and materials for selecting for and/or utilizing particulardesirable traits of functional ligands in a multiplexed manner, and moreparticularly to methods and materials for selecting for and/or utilizingparticular structural changes of functional ligands in a multiplexedmanner.

SEQUENCE LISTING

Deoxyribonucleic acid (DNA) sequences, which are disclosed in the ASCIItext file entitled “P1011US18_ST25.txt”, created on Jan. 30, 2019 and of78.5 KB in size, which is incorporated by reference in its entirety,herein are intended to include other aptamers incorporatingmodifications, truncations (e.g. trivial truncations, such as 1-5nucleotides removed at an end, which consist essentially of the samesequence and retains binding to the target molecule), incorporationsinto larger molecules or complexes (e.g. the aptamer sequence within alonger nucleic acid strand), and/or other aptamers having substantialstructural or sequence homology, for example, greater than 75% sequencehomology within a similar length of nucleic acid (e.g. similar to within5-10 nucleotides in length with significant sequence homology withinthat length, such as greater than 75%), as well as RNA and/or othernon-DNA/RNA aptamers. The disclosed aptamers may also bind to homologousproteins or molecules from organisms other than the organisms listedherein, to recombinant or non-recombinant versions of the proteins ormolecules, to modified versions of the proteins or molecules, toproteins or molecules from sources other than the source listed herein.The aptamers are artificial, non-naturally occurring sequences designedand/or selected for specific and/or high affinity binding to a targetmolecule, such as, without limitation, SEQ ID Nos. 1-69 may generallybind to the target molecule lipoarabinomannan (LAM), SEQ ID Nos. 70-188may generally bind to the target molecule Tetrakis (hydroxymethyl)phosphonium sulfate (THPS), and SEQ ID Nos. 189-301 may generally bindto the target molecule bronopol. SEQ ID No. 302 and SEQ ID No. 304 maygenerally be utilized as a leading or priming sequence appended to the5′-end of any of the aptamer sequences presented herein and SEQ ID No.303 and SEQ ID No. 305 may generally be utilized as a trailing orpriming sequence appended to the 3′-end of any of the aptamer sequencespresented herein, or vice versa or any combination thereof.

BACKGROUND OF THE INVENTION

Aptamers, which are nucleic acid ligands capable of binding to moleculartargets, have recently attracted increased attention for their potentialapplication in many areas of biology and biotechnology. They may be usedas sensors, therapeutic tools, to regulate cellular processes, as wellas to guide drugs to their specific cellular target(s). Contrary to theactual genetic material, their specificity and characteristics are notdirectly determined by their primary sequence, but instead by theirsecondary and/or tertiary structure. Aptamers have been recentlyinvestigated as immobilized capture elements in a microarray format.Others have recently selected aptamers against whole cells and complexbiological mixtures. Aptamers may also, for example, exhibit changes intheir secondary and/or tertiary structure depending on whether it iscomplexed or uncomplexed with a target molecule.

Aptamers are commonly identified by an in vitro method of selectionsometimes referred to as Systematic Evolution of Ligands by EXponentialenrichment or “SELEX”. SELEX typically begins with a very large pool ofrandomized polynucleotides which is generally narrowed to one aptamerligand per molecular target. Once multiple rounds (typically 10-15) ofSELEX are completed, the nucleic acid sequences are identified byconventional cloning and sequencing. Aptamers have most famously beendeveloped as ligands to important proteins, rivaling antibodies in bothaffinity and specificity, and the first aptamer-based therapeutics arenow emerging. More recently, however, aptamers have been also developedto bind small organic molecules and cellular toxins, viruses, and eventargets as small as heavy metal ions.

SUMMARY OF THE INVENTION

The present invention relates to methods and materials for multiplexedutilization of collections of functional ligands, particularly tomethods and materials for selecting for and/or utilizing particulardesirable traits of functional ligands in a multiplexed manner, and moreparticularly to methods and materials for selecting for and/or utilizingparticular structural changes of functional ligands in a multiplexedmanner.

In general, functional ligands may be selected for and/or utilized fortheir ability to bind or complex to particular target molecules, and/orfor the manner in which they bind or complex to particular targetmolecules, such as by exhibiting a detectable structural change. Forexample, numerous biomolecules, such as nucleic acids and peptides, takeon varied secondary and tertiary structures in response to differentenvironments or by associating with other molecules. Functional ligandsmay generally include biomolecules such as nucleic acids, such assingle-stranded nucleic acids and double-stranded nucleic acids orcombinations or regions of both, peptides, other biopolymers and/orcombinations or modifications thereof, such as artificially modifiednucleic acids, synthetic analogs and the like. A collection of thesefunctional ligands may be utilized for various purposes, which mayinclude, but are not limited to, selecting members of the collection forbinding activity to particular target molecule(s) which may result in adesired structural change of the binding member(s), utilizing acollection of functional ligands to detect and/or quantify the presenceor absence of target molecule(s) in a sample, determining whethermembers of a collection bind to more than one target molecule or whetherbinding events are affected by the presence of multiple targetmolecules, and/or any other appropriate purpose for utilizing suchcollections. The target molecule(s) may be, for example and withoutlimitation, proteins, cells, small molecules, biomolecules, and/orcombinations or portions thereof.

In general, the collection may be present in a spatial arrangement, suchas an array, collection of discrete droplets or multiwell plate, orother system where particular locations of functional ligands are knownor readily determinable, such as with tagged or labeled beads. Forexample, the functional ligands may be arrayed in a stable spatialarrangement or they may be tagged or marked in a manner that theirparticular locations are determinable (discriminating between differentfunctional ligands). Structural changes may be directly observable, suchas through microscopy, microscale thermophoresis (MST), backscatteringinterferometry (BSI), and/or any other appropriate observation method.Structural changes may also be detectable through secondary events, suchas by detecting changes in fluorescence or other radiation emissions dueto alterations in the structure of a functional ligand.

In one aspect of the present invention, a method for selectingfunctional ligands may include providing a collection of functionalligands, introducing at least one target molecule and detecting possiblebinding activity between at least one member of the collection and theat least one target molecule by detecting a structural change in the atleast one member. In some embodiments, the functional ligands may bindto or complex with another molecule, which may serve as an indicator, ina manner that is affected by a binding event between the functionalligand and a target molecule. For example, the binding of the functionalligand and the indicator may be disrupted by the binding of a targetmolecule to the functional ligand, or vice versa. The indicator may alsobe a part of the functional ligand, such as forming a different regionof the functional ligand that may self-associate.

The functional ligand and the indicator may each carry a label or tagthat may interact with each other to produce a detectable signal, orinteract with each other to reduce a detectable signal. For example, thefunctional ligand and the indicator may carry a radiation-emitting labeland a radiation-quenching or reducing label, respectively, or viceversa. They may also carry a pair of labels that interact via Försterresonance energy transfer (FRET) and/or any other appropriatesignal-interaction mechanism.

In general, variations to the binding conditions may also be employed,such as to detect variations in structural changes, binding affinity,cross-reactivity, detection limits, and/or any other appropriatevariation. For example variations in binding conditions may include, butare not limited to, concentrations of the target molecules, inclusion ofother target molecules, variation in pH, temperature, pressure, flow,electrical gradient, solvent, degree of complementarity betweenfunctional ligands and indicators, solute makeup/concentration, spacingof functional ligands, spacing between functional ligands and asubstrate, and/or any other appropriate variation in binding conditions.

In some embodiments, the functional ligands included in a collection maybe randomized or unknown. In other embodiments, at least one of thefunctional ligands may be selected previously for a known or suspectedtrait or characteristic, such as known binding to a particular targetmolecule, predicted or observed structural changes during bindingevents, and/or any other known or suspected trait or characteristic.This may be desirable, for example, to efficiently utilize prior data orexperimental results to speed up or narrow selection.

In some exemplary embodiments, the functional ligands may include asingle-stranded nucleic acid, such as an aptamer, which may hybridizewith an indicator single-stranded nucleic acid in a manner that isaffected by the presence of a target molecule of the functional ligand.In some embodiments, the indicator nucleic acid or “Signal oligo” mayhybridize to a nucleic acid aptamer in the absence of a target moleculeand be displaced and/or occluded from hybridizing upon binding of atarget molecule to the aptamer. In some embodiments, the Signal oligoand the target molecule may bind to the same region (or part thereof) ofthe aptamer which may result in competitive binding between them. Inother embodiments, the aptamer may adopt a conformational change when itbinds to the target molecule, which may, without being bound to anyparticular theory, result in the occlusion of the Signal oligo orotherwise making the hybridization thermodynamically unfavorable due tothe conformation change. The unhybridized Signal oligo and/or aptamermay then be utilized to detect a structural change in the aptamer.

In some embodiments, the detected structural change at a particularspatial location or with a particular determinable location may beutilized to indicate which functional ligand experienced a binding eventin order to correlate whether a particular functional ligand binds to aparticular target molecule.

In some embodiments, multiple target molecules may be introduced todetermine which, if any, of the collection bind to them. For example, acollection of potentially binding functional ligands may be exposed to atarget molecule and then a detection may be performed to determine ifany of the collection binds. Then another target molecule may be addedand a further detection performed and so forth. In general, the knownspatial locations of particular functional ligands may be known ordeterminable for each exposure/binding with a known target molecule suchthat binding events may be correlated to particular functional ligandsand target molecules.

In another aspect of the present invention, a method for utilizingfunctional ligands may include providing a collection of functionalligands which are known to bind to particular target molecules in amanner that produces a detectable structural change, introducing asample to such collection and determining whether such sample containsany target molecules of such collection of functional ligands bydetecting any structural changes in such functional ligands. In someembodiments, the functional ligands may bind to or complex with anothermolecule, which may serve as an indicator, in a manner that is affectedby a binding event between the functional ligand and a target molecule.For example, the binding of the functional ligand and the indicator maybe disrupted by the binding of a target molecule to the functionalligand, or vice versa. The indicator may also be a part of thefunctional ligand, such as forming a different region of the functionalligand that may self-associate.

In some exemplary embodiments, the functional ligand may include anaptamer, as discussed above, which may associate with a Signal oligo.The unhybridized Signal oligo and/or aptamer may then be utilized todetect a structural change in the aptamer.

In some embodiments, the detected structural change at a particularspatial location or with a particular determinable location may beutilized to indicate which functional ligand experienced a binding eventand thus which target molecule(s) are present in the sample.

The degree of detected structural changes may also be utilized to, forexample, to determine abundance and/or concentration of a targetmolecule(s) in the sample.

In some embodiments, functional ligands may be predisposed on an arraysubstrate in a predetermined spatial arrangement. The functional ligandsmay, for example, be covalently or otherwise attached to the substrate.The indicators, such as the Signal oligos, may also be attached to thesubstrate rather than the functional ligands to which they bind orhybridize, or both may be attached in a way that they may bind orhybridize and also produce a detectable structural change when exposedto an appropriate target molecule.

In further embodiments, functional ligands and/or the indicators may besynthesized in situ on the array, such as by light directed in situnucleic acid synthesis.

The members of the collection and/or the indicators may also includedetectable portions, such as, for example, fluorescent moieties,radioactive tags and/or other appropriate detectable portions.

In some embodiments, any displaced functional ligands and/or indicatorsmay be collected and/or otherwise subjected to a sequencing and/orcompositional analysis, such as to verify which functional ligands, ifany, experienced a binding event that resulted in a free functionalligand and/or indicator that may be analyzed.

In further embodiments, a collection of functional ligands may includepeptide sequences and contacting the collection with at least one targetmolecule. In some exemplary embodiments, the peptide sequence may betagged, linked, marked and/or otherwise associated with a nucleic acidsequence. The nucleic acid sequence may be, for example, representativeof the sequence of the peptide. For example, the nucleic acid maysubstantially encode the peptide sequence. Also for example, the nucleicacid may be a unique or semi-unique identifier sequence. The nucleicacid sequence may then be utilized to bind an indicator, as describedabove, such that a peptide bound to a target molecule may be correlatedby the structural change in the nucleic acid sequence and/or theindicator.

The present invention together with the above and other advantages maybest be understood from the following detailed description of theembodiments of the invention and as illustrated in the drawings. Thefollowing description, while indicating various embodiments of theinvention and numerous specific details thereof, is given by way ofillustration and not of limitation. Many substitutions, modifications,additions or rearrangements may be made within the scope of theinvention, and the invention includes all such substitutions,modifications, additions or rearrangements.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1, 1 a, 1 b and 1 c illustrate an embodiment of a functionalligand attached to a substrate and indicator interacting with thepresence of a target molecule to produce a signal;

FIGS. 2, 2 a, 2 b and 2 c illustrate embodiments of a functional ligandattached to a substrate interacting with the presence of a targetmolecule to produce a signal;

FIGS. 3, 3 a and 3 b illustrates an embodiment of a functional ligandand an indicator attached to a substrate interacting with the presenceof a target molecule to produce a signal;

FIG. 4 illustrates a layout of a microfluidic chip with a pump and acollection vessel;

FIG. 4a illustrates the generation of discrete droplets containingfunctional ligands;

FIG. 5 shows an imaging capture from a microfluidic chip showingfluorescence from particular locations due to target molecule binding;

FIGS. 6 and 6 a illustrates an embodiment of a functional ligand and anindicator attached to a substrate with a spacing region interacting withthe presence of a target molecule to produce a signal;

FIG. 7 shows the fluorescence emissions from an example of theembodiment in FIGS. 6 and 6 a with target molecule THPS; and

FIG. 8 shows the fluorescence emissions from an example with the targetmolecule bronopol.

DETAILED DESCRIPTION OF THE INVENTION

The detailed description set forth below is intended as a description ofthe presently exemplified methods, devices, and compositions provided inaccordance with aspects of the present invention and is not intended torepresent the only forms in which the present invention may be practicedor utilized. It is to be understood, however, that the same orequivalent functions and components may be accomplished by differentembodiments that are also intended to be encompassed within the spiritand scope of the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devicesand materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the exemplifiedmethods, devices and materials are now described.

The present invention relates to methods and materials for multiplexedutilization of collections of functional ligands, particularly tomethods and materials for selecting for and/or utilizing particulardesirable traits of functional ligands in a multiplexed manner, and moreparticularly to methods and materials for selecting for and/or utilizingparticular structural changes of functional ligands in a multiplexedmanner.

In general, functional ligands may be selected for and/or utilized fortheir ability to bind or complex to particular target molecules, and/orfor the manner in which they bind or complex to particular targetmolecules, such as by exhibiting a detectable structural change. Forexample, numerous biomolecules, such as nucleic acids and peptides, takeon varied secondary and tertiary structures in response to differentenvironments or by associating with other molecules. Further in general,functional ligands may generally include any molecule that undergoeslarge or otherwise detectable conformational or structural changes whenparticular binding events occur.

Functional ligands may generally include biomolecules such as nucleicacids, such as single-stranded nucleic acids and double-stranded nucleicacids or combinations or regions of both, peptides, other biopolymersand/or combinations or modifications thereof, such as artificiallymodified nucleic acids, synthetic analogs and the like. Non-naturallyoccurring sequences of functional ligands, such as nucleic acids andnucleic acid analogs, such as aptamers, may also be useful byinteracting with a target molecule in a manner not present in naturallyoccurring systems or situations, such as by, for example, not beingalready present or having a pre-existing function in a naturallyoccurring setting.

Other examples of functional ligands may include, but are not limitedto, G-protein receptors, ion channels, promoter or enhancer elements ofDNA, nucleic acid beacons/probes which exhibit conformational changes,and/or any other appropriate functional ligands with the desiredstructural/conformational changing properties.

In general, functional ligands may generally include nucleic acids,particularly single-stranded nucleic acids, peptides, other biopolymersand/or combinations or modifications thereof. Nucleic acid sequences mayinclude naturally-occurring biomolecules such as ribonucleic acid (RNA),deoxyribonucleic acid (DNA), artificially modified nucleic acids, and/orcombinations thereof. In general, modified nucleic acid bases may beutilized and may include, but are not limited to,2′-Deoxy-P-nucleoside-5′-Triphosphate, 2′-Deoxyinosine-5′-Triphosphate,2′-Deoxypseudouridine-5′-Triphosphate, 2′-Deoxyuridine-5′-Triphosphate,2′-Deoxyzebularine-5′-Triphosphate,2-Amino-2′-deoxyadenosine-5′-Triphosphate,2-Amino-6-chloropurine-2′-deoxyriboside-5′-Triphosphate,2-Aminopurine-2′-deoxyribose-5′-Triphosphate,2-Thio-2′-deoxycytidine-5′-Triphosphate,2-Thiothymidine-5′-Triphosphate, 2′-Deoxy-L-adenosine-5′-Triphosphate,2′-Deoxy-L-cytidine-5′-Triphosphate,2′-Deoxy-L-guanosine-5′-Triphosphate,2′-Deoxy-L-thymidine-5′-Triphosphate, 4-Thiothymidine-5′-Triphosphate,5-Aminoallyl-2′-deoxycytidine-5′-Triphosphate,5-Aminoallyl-2′-deoxyuridine-5′-Triphosphate,5-Bromo-2′-deoxycytidine-5′-Triphosphate,5-Bromo-2′-deoxyuridine-5′-Triphosphate,5-Fluoro-2′-deoxyuridine-5′-Triphosphate,5-Trifluoromethyl-2-deoxyuridine-5′-Triphosphate, and/or any otherappropriate modified nucleic acid base. It may generally be understoodthat the nucleoside triphosphates (NTPs) listed above may generallyrefer to any appropriate phosphate of the modified base, such asadditionally, for example, monophosphates (NMPs) or diphosphates (NDPs)of the base. Embodiments of the SELEX method may generally be utilizedto select or preselect for aptamers to be used in a collection. Thebasic SELEX protocol and aptamers are described in U.S. Pat. No.5,270,163, entitled “Methods for identifying nucleic acid ligands,” theentire contents of which are hereby incorporated by reference.

A collection of these functional ligands may be utilized for variouspurposes, which may include, but are not limited to, selecting membersof the collection for binding activity to particular target molecule(s)which may result in a desired structural change of the bindingmember(s), utilizing a collection of functional ligands to detect and/orquantify the presence or absence of target molecule(s) in a sample,determining whether members of a collection bind to more than one targetmolecule or whether binding events are affected by the presence ofmultiple target molecules, and/or any other appropriate purpose forutilizing such collections. The target molecule(s) may be, for exampleand without limitation, proteins, cells, small molecules, biomolecules,and/or combinations or portions thereof.

In general, the collection may be present in a spatial arrangement, suchas an array (e.g. microarrays, microfluidic chips (i.e. microchips),handspotted arrays, etc.), collection of discrete droplets or multiwellplate, or other system where particular locations of functional ligandsare known or readily determinable, such as with tagged or labeled beads.For example, the functional ligands may be arrayed in a stable spatialarrangement or they may be tagged or marked in a manner that theirparticular locations are determinable (discriminating between differentfunctional ligands). In other examples, the functional ligands may beattached to beads or other movable substrates which may then be disposedin known locations or spatially organized in a manner where differentsets of beads/movable substrates may be kept separated for sorting,selective manipulation and/or prevention of cross-talk/interference fromneighboring beads/movable substrates. For example, the beads/movablesubstrates may be present in separate wells of multiwell plates or inseparate discrete droplets.

In embodiments utilizing an array with a solid substrate, the substratesused may be glass, ceramic, metal or polymeric, and/or any otherappropriate material. In general, it may be desirable to utilize amaterial that is convenient for attaching functional ligands and/or forin situ synthesis. Polymers may include synthetic polymers as well aspurified biological polymers. The substrate may also be any film, whichmay be non-porous or macroporous.

The substrate may be generally planar and may be of any appropriategeometry such as, for example, rectangular, square, circular,elliptical, triangular, other polygonal shape, irregular and/or anyother appropriate geometry. The substrate may also be of other forms,such as cylindrical, spherical, irregular and/or any other appropriateform.

Appropriate ceramics or metals may include, for example, hydroxyapatite,alumina, graphite, graphene, buckyballs, silica, gold, silver, andpyrolytic carbon.

Appropriate synthetic materials may include polymers such as polyamides(e.g., nylon), polyesters, polystyrenes, polyacrylates, vinyl polymers(e.g., polyethylene, polytetrafluoroethylene, polypropylene andpolyvinyl chloride), polycarbonates, polyurethanes, poly dimethylsiloxanes, cellulose acetates, polymethyl methacrylates, ethylene vinylacetates, polysulfones, nitrocelluloses and similar copolymers. Thesesynthetic polymers may be woven or knitted into a mesh to form a matrixor similar structure. Alternatively, the synthetic polymer materials canbe molded or cast into appropriate forms.

Biological polymers may be naturally occurring or produced in vitro byfermentation and the like or by recombinant genetic engineering.Recombinant DNA technology can be used to engineer virtually anypolypeptide sequence and then amplify and express the protein in eitherbacterial or mammalian cells. Purified biological polymers can beappropriately formed into a substrate by techniques such as weaving,knitting, casting, molding, extrusion, cellular alignment and magneticalignment. Suitable biological polymers include, without limitation,collagen, elastin, silk, keratin, gelatin, polyamino acids,polysaccharides (e.g., cellulose and starch) and copolymers thereof.

Any suitable substrate may be susceptible to adhesion, attachment oradsorption by functional ligands or indicators, as appropriate. Thesusceptibility may be inherent or modified. In one example, the surfacesof substrates may be susceptible to adhesion, attachment or adsorptionto nucleic acids or peptides/proteins. In another example, the surfacesof substrates may be susceptible to adhesion, attachment or adsorptionto proteins or peptides and not to nucleic acids, or vice versa.

In one exemplary embodiment, a glass substrate may have a layer orcoating of a material that promotes adhesion with targets, such asproteins, peptides or nucleic acids, materials that maybe charged, suchas those that are positively charged, for binding target materials.Examples of charged materials include cellulosic materials, for example,nitrocellulose, methylcelluose, ethylcellulose, carboxymethyl cellulose,hydroxyethyl cellulose, methylhydroxypropyl cellulose; epoxies, PVDF(polyvinylidene fluoride); partially or fully hydrolyzed poly(vinylalcohol); poly(vinylpyrrolidone); poly(ethyloxazoline); poly(ethyleneoxide)-co-poly(propylene oxide) block copolymers; polyamines;polyacrylamide; hydroxypropylmethacrylate; polysucrose; hyaluronic acid;alginate; chitosan; dextran; gelatin and mixtures and copolymersthereof.

In other embodiments, if the substrate is not susceptible for attachmentby charged materials, or may be susceptible only for attachment bywrongly charged materials, some areas of the substrate may haveadhesives, binding agents, or similar attached, adsorbed or coatedthereon. Examples of adhesives may include any suitable adhesives thatbind the charged materials.

The functional ligands may be present on the substrate discretely or inclusters. The distance between the discrete functional ligands may beclose or may be far apart and may usually be of different functionalligands. Clusters may be used for multiple spots of a single functionalligand. In general, the distance between placements may be chosen to aidin preventing direct interactions between adjacent functional ligands,to aid in preventing unwanted multiple binding events between a targetmolecule and adjacent functional ligands, to aid in preventinginterference of a binding event due to proximity to an adjacentfunctional ligand and/or in preventing any other applicable unwantedinteractions.

In one embodiment, the substrate may be macroporous. Macroporoussubstrates may be desirable, for example, if the different functionalligands are very close together. Closely packed functional ligands may,for example, increase the efficiency of the utilization of a particularsubstrate. A macroporous substrate may be suited for balancing betweenefficiency and separation. For a macroporous substrate, the walls of thepores may be sufficient to separate even closely packed functionalligands if the pores are large enough to enable the binding process tooccur within the pores.

Also, for macroporous substrates, the pores may have an average diametergreater than the average size of the target molecule(s) such that theymay enter or partly enter the pores for binding events to occur.Hydrogels may also be useful for binding or anchoring functional ligandsto the pores. Hydrogels may also fill the pores under fluid conditionsand present a smooth surface for fluid flow while at the same time maykeep the fluid from flowing through the pores.

The plurality of functional ligands may be arranged in any appropriatemanner such as, for example, in circular or elliptical spots, square orrectangular spots, stripes, concentric rings and/or any otherappropriate arrangement on the subject.

In some embodiments, the functional ligands may also be disposed onbeads or other free-floating substrates. For example, glass beads,agarose or cellulosic beads (e.g. Sepharose™ or Sephadex™)nanomaterials, nanoparticles and/or any other appropriate free floatingsubstrate may be utilized. The beads or free-floating substrates mayalso generally be labeled or tagged such that the identity of thefunctional ligands attached to them are determinable by identifying thelabel or tag. For example, fluorescently coded labels (such as colorcombination fluors) may be utilized to barcode or otherwiseuniquely/semi-uniquely identify particular bead(s).

Biotin may also be included on functional ligands or indicator moleculesto allow them to be attached to a substrate, such as substrates coatedwith streptavidin or similar molecules.

Structural changes may be directly observable, such as throughmicroscopy, microscale thermophoresis (MST), backscatteringinterferometry (BSI), and/or any other appropriate observation method.Structural changes may also be detectable through secondary events, suchas by detecting changes in fluorescence or other radiation emissions dueto alterations in the structure of a functional ligand.

Pairs of corresponding chromophores and/or fluorophores may be utilizedfor their ability of altering the conversion efficiency of the radiationconverting chromophore and/or fluorophores. For example, converting andabsorbing chromophore and/or fluorophores pairs in the radiation portionof interest may include, but are not limited to: ALEXA633™/QSY21™,CY5υ/QSY21™, ALEXA647™/QSY21™, ALEXA647™/ALEXA680™,ALEXA680™/allophycocyanin (APC), ALEXA700™/APC, CY3™/BHQ-2, and/orALEXA750™/APC (Molecular Probes, Inc.), and/or any other appropriatechromophore and/or fluorophores pair. It is contemplated, however, thatany suitable pair of first and/or second radiation convertingchromophore and/or fluorophore and radiation absorbing chromophoreand/or fluorophore, whether now known or later developed, is within thescope of the present invention.

A chromophore-quencher pair may also include a metallic quenchingelement, such as a gold or other metallic substrate.

In one aspect of the present invention, a method for selectingfunctional ligands may include providing a collection of functionalligands, introducing at least one target molecule and detecting possiblebinding activity between at least one member of the collection and theat least one target molecule by detecting a structural change in the atleast one member. In some embodiments, the functional ligands may bindto or complex with another molecule, which may serve as an indicator, ina manner that is affected by a binding event between the functionalligand and a target molecule. For example, the binding of the functionalligand and the indicator may be disrupted by the binding of a targetmolecule to the functional ligand, or vice versa.

Not all binding events may necessarily result in structural orconformational changes which may be detected. Thus, these methods may beutilized to distinguish between structural/conformational switchingfunctional ligands and binding functional ligands which do not undergosuch switching behavior. In some situations, structural/conformationalswitching functional ligands may be more preferable or desirable to usein particular applications as opposed to functional ligands which do notundergo structural/conformational switching, such as where thestructural/conformational switching is more easily detected orquantified as opposed to non-switching binding events. Additionally,some switching events may enable or aid in “one step” detection methods,such as for example with radiation emitting methods as described below,rather than binding events which do not exhibit switching behavior whichmay require additional steps to detect binding.

FIG. 1 illustrates an example of a functional ligand 100 and anindicator 200, shown with the functional ligand 100 attached to asubstrate 90. The functional ligand 100 may generally include a targetmolecule binding region 102, an indicator binding region 104 and/or alinker region 106, where the linker region 106 may serve to space therest of the functional ligand 100 from the substrate 90 such that it mayinteract freely with other parts of the system, such as a targetmolecule 80 and/or the indicator 200. The target molecule binding region102 and the indicator binding region 104 may also be, for example,overlapping to some degree, spaced apart by another region, onecontained within the other and/or any other desirable arrangement. Asillustrated in FIG. 1A, indicator 200 may generally bind to theindicator binding region 104, such as by hybridization or otherreversible binding. In general, the indicator 200 may bind specificallyto an indicator binding region 104 of one species of functional ligand100 within a collection, or it may bind to multiple or all of themembers of a collection in a non-specific manner. A non-specificindicator 200 may be desirable for ease and/or cost savings forproducing the indicators, as well as for consistency in designing of thefunctional ligands 100.

In the presence of a target molecule 80 which binds to the functionalligand 100, the functional ligand 100 may generally adopt a conformationwhich binds to the target molecule 80, as illustrated in FIG. 1B. Incases where the structural change of the functional ligand 100 issignificant, the conformational change may generally result in theindicator 200 being displaced and/or otherwise unable to remain bound tothe functional ligand 100, as illustrated in FIG. 1B. This may generallybe detected as a signal or observable event A.

The indicator may also be a part of the functional ligand, such asforming a different region of the functional ligand that mayself-associate. FIG. 2 illustrates an example of a functional ligand100′ which self-binds or self-hybridizes at zone 104′ which may bedisrupted to produce a signal or observable event A when the functionalligand 100′ binds a target molecule 80, as shown in FIG. 2A.

The functional ligand and the indicator may each carry a label or tagthat may interact with each other to produce a detectable signal, orinteract with each other to reduce a detectable signal. For example, thefunctional ligand and the indicator may carry a radiation-emitting labeland a radiation-quenching or reducing label, respectively, or viceversa. They may also carry a pair of labels that interact via Försterresonance energy transfer (FRET) and/or any other appropriatesignal-interaction mechanism.

FIG. 1C illustrates the example shown in FIGS. 1, 1A and 1B where thefunctional ligand 100 and the indicator 200 carry a signal interactingpair, such as a signal emitting label 112 and a signal attenuating label210, respectively, such that when the functional ligand 100 and theindicator 200 are bound, the labels 112, 210 interact due to proximityresulting in the signal B from label 112 being attenuated or quenched.The binding of the target molecule 80 and the subsequent increase indistance between the labels 112, 210 may then result in the emission ofa signal B from the label 112 which may be detected.

FIGS. 2B and 2C illustrate the example shown in FIGS. 2 and 2A where thefunctional ligand 100′ carries a signal emitting label 112 and a signalattenuating label 110 at different positions of the functional ligand100′ such that when the self-interaction 104′ occurs, the labels 112,110 interact due to proximity resulting in the signal B from label 112being attenuated or quenched, or vice versa. The binding of the targetmolecule 80 and the subsequent increase in distance between the labels112, 110 may then result in the emission of a signal B from the label112 (or 110 as appropriate) which may be detected.

FIGS. 6 and 6 a illustrate an example where a functional ligand 400 andan indicator 300 carry a signal interacting pair, such as a signalemitting label 405 and a signal attenuating label 305, respectively (orvice versa), such that when the functional ligand 400 and the indicator300 are bound, such as through hybridization D of complementary orpartially complementary regions 302, 402, the labels 305, 405 interactdue to proximity resulting in the signal B from label 405 (or 305 ifreversed) being attenuated or quenched. The binding of the targetmolecule 80 to the binding region 410 of the functional ligand 400, asillustrated in FIG. 6 a, may generally cause a conformational change inthe functional ligand 400 which may generally result in thecomplementary or partially complementary regions 302, 402 dehybridizingE to release the functional ligand 400 from the indicator 300 and thesubsequent increase in distance between the labels 305, 405 may thenresult in the emission of a signal B which may be detected. The labels305, 405 may be attached to or integrated into the functional ligand 400and the indicator 300, such as by covalent attachment, within the strandof the molecule, such as in the middle (as illustrated with label 305 inthe middle of indicator 300) or at a terminal end (e.g. the 5′ end 401as illustrated or the 3′ end 403 if the region 404 is designed tohybridize with the region 302). Other attachment methods may include butare not limited to, non-covalent bonding, streptavidin-biotin couplingand/or any other appropriate method.

In general, the functional ligands and indicators may also be utilizedin solution or otherwise suspending a fluid without an attachment to asubstrate. To aid in separating the labels from the functional ligandsand indicators that have dehybridized due to target, separationtechniques may be utilized, such as separating based on size, mass,charge, or other differing characteristics between the functionalligands and indicators. One of the functional ligands or the indicatorsmay also be attached to a feature which aids in separation, such as amagnetic bead or other appropriate feature or substrate, such that oneof the functional ligands or the indicators (when unhybridized) may bepulled away from the other, such as by applying a magnetic field and/orwashing away the non-magnetically attached molecules. In general, it maybe desirable to aid in separating the unhybridized functional ligandsand indicators such there may be appropriate distance between theirlabels to produce detectable signal.

In general, variations to the binding conditions may also be employed,such as to detect variations in structural changes, binding affinity,cross-reactivity, detection limits, and/or any other appropriatevariation. For example variations in binding conditions may include, butare not limited to, concentrations of the target molecules, inclusion ofother target molecules, variation in pH, temperature, pressure, flow,electrical gradient, solvent, degree of complementarity betweenfunctional ligands and indicators, solute makeup/concentration, spacingof functional ligands, spacing between functional ligands and asubstrate, and/or any other appropriate variation in binding conditions.

In some embodiments, as illustrated in FIGS. 6 and 6 a, a spacing regionmay be provided between the substrate and a label on the indicator, asshown with spacing region 304 providing a gap between the substrate 90and the label 305. This may be desirable, for example, to space thelabel 305 away from the substrate 90 to aid in preventing interactionsbetween the label 305 and the substrate 90 or the molecular couplingbetween the substrate 90 and the indicator 300 which may, for exampleand without being bound to any particular theory, cause adverse effectson the signal emission of label 305. The spacing region may also, forexample and without being bound to any particular theory, space theregion 302 away from the substrate 90 to aid in preventing interactionsbetween the substrate 90 and the region 302 which may interfere withproper hybridization D between the regions 302, 402. In general, thespacing region may be of any appropriate length, such as at least 5nucleotides in length and generally up to about 20 nucleotides inlength.

In some embodiments, the functional ligands included in a collection maybe randomized or unknown. In other embodiments, at least one of thefunctional ligands may be selected previously for a known or suspectedtrait or characteristic, such as known binding to a particular targetmolecule, predicted or observed structural changes during bindingevents, and/or any other known or suspected trait or characteristic.This may be desirable, for example, to efficiently utilize prior data orexperimental results to speed up or narrow selection.

In some exemplary embodiments, the functional ligands may include asingle-stranded nucleic acid, such as an aptamer, which may hybridizewith an indicator single-stranded nucleic acid in a manner that isaffected by the presence of a target molecule of the functional ligand.In some embodiments, the indicator nucleic acid or “Signal oligo” mayhybridize to a nucleic acid aptamer in the absence of a target moleculeand be displaced and/or occluded from hybridizing upon binding of atarget molecule to the aptamer. In some embodiments, the Signal oligoand the target molecule may bind to the same region (or part thereof) ofthe aptamer which may result in competitive binding between them. Inother embodiments, the aptamer may adopt a conformational change when itbinds to the target molecule, which may, without being bound to anyparticular theory, result in the occlusion of the Signal oligo orotherwise making the hybridization thermodynamically unfavorable due tothe conformation change. The unhybridized Signal oligo and/or aptamermay then be utilized to detect a structural change in the aptamer.

In general, the Signal oligo may be of sufficient length andcomplementarity to hybridize to the aptamer serving as a functionalligand. The length and complementarity (e.g. hybridization mismatches)may also be varied or modified to alter the melting temperature (Tm) ofthe hybridization and thus the relative strength of the hybridization.This may be desirable to tune the degree of structural change of theaptamer necessary to cause dehybridization of the Signal oligo, whichmay thus be utilized in tuning the affinity of the aptamer:targetmolecule interactions that are detected. In general, the Signal oligomay be approximately 6 nucleotides long or longer, and/or anyappropriate length that may effectively hybridize at the temperatureand/or other environmental conditions present in the system, as shorterlengths may not provide a sufficiently favorable thermodynamichybridization.

In some embodiments, the detected structural change at a particularspatial location or with a particular determinable location may beutilized to indicate which functional ligand experienced a binding eventin order to correlate whether a particular functional ligand binds to aparticular target molecule.

In some embodiments, multiple target molecules may be introduced todetermine which, if any, of the collection bind to them. For example, acollection of potentially binding functional ligands may be exposed to atarget molecule and then a detection may be performed to determine ifany of the collection binds. Then another target molecule may be addedand a further detection performed and so forth. In general, the knownspatial locations of particular functional ligands may be known ordeterminable for each exposure/binding with a known target molecule suchthat binding events may be correlated to particular functional ligandsand target molecules.

In further embodiments, functional ligands and/or the indicators may besynthesized in situ on the array, such as by light directed in situnucleic acid synthesis or by printing peptide or protein expressinggenes onto a surface and using cell-free peptide/protein expression andcapture to synthesize peptides/proteins at predetermined locations on asurface. This may be desirable to control the position of eachfunctional ligand and/or indicator.

Presynthesized functional ligands and/or indicators may also be attachedto predetermined locations on an array, such as with ligation reactionsand/or any other appropriate method.

In some embodiments, the functional ligands and/or the indicators mayalso be placed into discrete fluid droplets or masses and disposed on asurface or array in a predetermined manner. For example, discrete fluiddroplets may be generated with one or a combination of functionalligands in each and placed or sputtered onto a surface, such as with theRaindance RainDrop™ technology, where the droplets are immiscible in acarrier fluid and thus are able to be kept separate and sorted/handledwithout the materials in each droplet interacting. In another example,fiber optic arrays may be utilized with a well formed on the end of eachfiber in a fiber optic bundle, thus enabling each individual well to belocalized by its carrier fiber and individually interrogated with light.For example, functional ligands and/or the indicators may be present onbeads or other small substrates which may be suspended within thedroplets. FIG. 4a illustrates an example of formation of discretedroplets where a stream 60 containing functional ligands and/orindicators may be carried in a bulk fluid stream 70 which flows D pastan interface 50 where streams of bulk fluid stream 70 exert a pinching Cin a pulsing fashion on the stream 60 to create individual droplets 62.

In another aspect of the present invention, a method for utilizingfunctional ligands may include providing a collection of functionalligands which are known to bind to particular target molecules in amanner that produces a detectable structural change, introducing asample to such collection and determining whether such sample containsany target molecules of such collection of functional ligands bydetecting any structural changes in such functional ligands. In someembodiments, the functional ligands may bind to or complex with anothermolecule, which may serve as an indicator, in a manner that is affectedby a binding event between the functional ligand and a target molecule.For example, the binding of the functional ligand and the indicator maybe disrupted by the binding of a target molecule to the functionalligand, or vice versa. The indicator may also be a part of thefunctional ligand, such as forming a different region of the functionalligand that may self-associate.

In some exemplary embodiments, the functional ligand may include anaptamer, as discussed above, which may associate with a Signal oligo.The unhybridized Signal oligo and/or aptamer may then be utilized todetect a structural change in the aptamer.

In some embodiments, the detected structural change at a particularspatial location or with a particular determinable location may beutilized to indicate which functional ligand experienced a binding eventand thus which target molecule(s) are present in the sample.

The degree of detected structural changes may also be utilized to, forexample, to determine abundance and/or concentration of a targetmolecule(s) in the sample.

In some embodiments, functional ligands may be predisposed on an arraysubstrate in a predetermined spatial arrangement. The functional ligandsmay, for example, be covalently or otherwise attached to the substrate.The indicators, such as the Signal oligos, may also be attached to thesubstrate rather than the functional ligands to which they bind orhybridize, or both may be attached in a way that they may bind orhybridize and also produce a detectable structural change when exposedto an appropriate target molecule.

FIG. 3 illustrates an example where the indicator 200′ is attached tothe substrate 90 and bound or hybridized to the functional ligand 100″via indicator binding region 104. In the presence of a target molecule80, the functional ligand 100″ may then unbind or dehybridize from theindicator 200″ to bind to the target molecule 80, such that anobservable signal or event A occurs, as shown in FIG. 3A, or in the casewith a pair of interacting labels 112, 210, the production of anobservable or detectable signal B, as shown in FIG. 3B.

The members of the collection and/or the indicators may also includedetectable portions, such as, for example, moieties or portions whichparticipate in detectable interactions or which produce detectablesignals, such as colorimetric interactions, refractive index changes,fluorescence interactions (e.g. Fluorescence Energy Transfer (FRET)),fluorescence enhancement upon moving to a different solvent environment,redox interactions, enzymatic interactions, pH reporting mechanisms,surface enhanced Raman scattering (SERS), isothermal DNA amplification,thermal or temperature changes, and/or any other appropriate detectableinteractions or signals.

In some embodiments, any displaced functional ligands and/or indicatorsmay be collected and/or otherwise subjected to a sequencing and/orcompositional analysis, such as to verify which functional ligands, ifany, experienced a binding event that resulted in a free functionalligand and/or indicator that may be analyzed. For example, unhybridizedor unbound functional ligands or indicators, as appropriate, may bewashed from the collection and sequenced or analyzed, such as by massspectrometry or other methods, to determine which functional ligandsexperienced a binding event that resulted in the dehybridization orunbinding. In general, it may be desirable that the indicators, ifutilized in this manner, be unique or semi-unique to a respectivefunctional ligand to aid in correlation. In general, aside from standardsequencing methods, parallel sequencing methods, such as, for example,massively parallel sequencing such as 454 Clonal Sequencing (Roche,Branford, Conn.), massively parallel clonal array sequencing, SolexaSequencing (Illumina, San Diego, Calif.), and/or any other appropriatesequencing method may be employed.

In some embodiments, the indicator, such as a Signal oligo, may serve asa primer in a nucleic acid amplification reaction, which may beperformed following contacting a collection of functional ligands withtarget molecule(s). This may be utilized to create amplificationproducts by extension of the indicator primers that are still hybridizedand not displaced, which may be detected, such as by addition of nucleicacid-binding dyes or probes.

In further embodiments, a collection of functional ligands may includepeptide sequences and contacting the collection with at least one targetmolecule. Examples of peptide or protein functional ligands may include,but are not limited to, G protein receptors, ion channels, antibodies,peptide aptamers, phage-displayed peptides, and mRNA-displayed peptides.In some exemplary embodiments, the peptide sequence may be tagged,linked, marked and/or otherwise associated with a nucleic acid sequence.The nucleic acid sequence may be, for example, representative of thesequence of the peptide. For example, the nucleic acid may substantiallyencode the peptide sequence. Also for example, the nucleic acid may be aunique or semi-unique identifier sequence. The nucleic acid sequence maythen be utilized to bind an indicator, as described above, such that apeptide bound to a target molecule may be correlated by the structuralchange in the nucleic acid sequence and/or the indicator.

In some embodiments, methods of incorporating and/or linking nucleicacids to peptides may be utilized, such as, for example, phage display,mRNA display, ribosome display, and/or any other appropriate method. Ingeneral, in phage display, a bacteriophage (phage) may be generated thatincludes a peptide sequence of interest in its protein coat. The phagemay further include a nucleic acid sequence that may be representativeof the peptide sequence within the nucleic acid of the phage. The phagemay then be contacted with target molecules. In general, in mRNAdisplay, a fusion product of a messenger RNA (mRNA) may be linked to apeptide that the mRNA encodes, such as with a puromycin-ended mRNA whichmay generally cause fusion of the mRNA to the nascent peptide in aribosome, which may then be contacted with target molecules. Also ingeneral, in ribosome display, a fusion product of a modified mRNA may beutilized that codes for a peptide, but lacks a stop codon and may alsoincorporate a spacer sequence which may occupy the channel of theribosome during translation and allow the peptide assembled at theribosome to fold, which may result in the peptide attached to theribosome and also attached to the mRNA. This product may then becontacted with target molecules. Other methods may include, but are notlimited to, yeast display, bacterial display, and/or any otherappropriate method.

Example of Multiplexed Selection of Aptamers with an Array

In an example of a selection protocol with an array, a microarray chipwith spatially organized spottings of functional ligands attached to thesurface of the chip (aptamer candidates with the sequences SEQ ID Nos.1-69 with 5′ leading sequence SEQ ID No. 302 and 3′ ending sequence SEQID No. 303), where the identity (e.g. sequence or other identifyinginformation) of the functional ligands at each spot is known, is used inconjunction with a fluidic delivery system, such as a syringe or pump,as illustrated in FIG. 4 with the microfluidic chip 300 with syringepump 310 and a collection tube 320 for capturing fluid that passesthrough the microfluidic chip 300. The example functional ligands wereselected to potential binding activity to a target molecule,lipoarabinomannan (LAM), a glycolipid, and a virulence factor associatedwith Mycobacterium tuberculosi. The functional ligands were thenattached to indicators, such as a Signal oligo which would be hybridizedto a nucleic acid aptamer functional ligand, in a buffer or othersolution to promote binding (e.g. 1 mM MgCl₂, 0.05% Tween-20, 1×PBS pH7.4 for hybridizing a Signal oligo to an aptamer), such as forapproximately 30 minutes. In an example, the aptamer candidates and theSignal oligos may be designed with a corresponding signal-interactingpair (e.g. a fluorophore and quencher pair or other pair as describedabove) such that upon hybridization, a baseline signal is generated(e.g. quenched fluorescence due to proximity). Signal oligos weredesigned to be approximately 7 bp in length and complementary to acorresponding portion of the aptamer candidates with a quencher (e.g. 5′Iowa Black RQ dark quencher). The background signal of the microchip wasmeasured to provide a baseline signal measurement. The microchip waswashed to remove excess Signal oligos with a buffer. LAM targetcontained in a buffer at 10 nM concentration was introduced to themicrochip and binding was allowed to occur for 30 minutes, after whichthe microchip was imaged with a GenePix 4000B at 532 nm. The bindingprocess was repeated with increasing concentrations of target (e.g. 50nM and 250 nM). FIG. 5 illustrates an example imaging of the microchiparray, with the brightness of the spots correlating to increased signaldetected. Without being bound to any particular theory, the increasedsignal is taken as correlating to greater conformational changing of theaptamer candidate due to dehybridization of the Signal oligo. SEQ IDNos. 31, 34, 35, 39, 41, 48 and 53 were identified from signal emittingspots on the microchip and determined to possess binding activity in theapproximately 500 pM affinity range to LAM.

Example of Detection of Signal from Addition of Target (THPS) toStructure-switching Aptamers and Indicators

In an example of detecting signal from structure switching aptamers andindicators when incubated with the target molecule of the aptamer, asubstrate coated with streptavidin was provided with indicator moleculesattached via biotin and including a spacing region of between 5 and 20nucleotides and then hybridized to an aptamer, as illustrated in FIG. 6with indicator 300 (single-stranded DNA) attached to substrate 90 withspacing region 304 and the aptamer 400 (single-stranded DNA) hybridizedD to it via regions 302, 402. The aptamer 400 was provided with a signalemitting label, as shown with label 405 (e.g. Cy3™ dye) attached at the5′-end of the aptamer 400. The indicator 300 was provided with a signalinteracting label 305 (e.g. BHQ-2 quencher), such that when hybridized,the signal interacting label 305 quenches emission of signal from thelabel 405 when exposed to excitation (e.g. 530 nm light). Progressiveconcentrations of target molecule 80 were then added to generateconformational change in the aptamer 400 (which was preselected as anaptamer binding to THPS from SEQ ID Nos. 70-188 flanked at its 5′-endwith SEQ ID No. 304 and at its 3′-end with SEQ ID 305) to causedehybridization or otherwise spacing from the indicator 300 to decreasequenching and increase emitted signal due to excitation from the signalemitting label 405. FIG. 7 illustrates the fluorescence detected (shownat 570 nm from a 530 nm excitation source) before addition of target(left bar), at the time of adding target (middle bar) and after 30minutes of incubation with target (right bar). As illustrated, aconcentration-dependent increase in fluorescence was observed withincreasing concentrations of target (from 0 to 5000 μM THPS).

Example of Detection of Signal from Addition of Target (Bronopol) toStructure-Switching Aptamers and Indicators

In an example of detecting signal from structure switching aptamers andindicators when incubated with the target molecule of the aptamer, asubstrate with an attached aptamer. The aptamer was provided with asignal emitting label as a fluorescent dye covalently attached to theaptamer. An indicator was provided with a signal interacting label as aquencher, such that when hybridized, the signal interacting label on theindicator quenches emission of signal from the label on the aptamer whenexposed to excitation (e.g. 530 nm light). Progressive concentrations oftarget molecule bronopol were then added to generate conformationalchange in the aptamer (which was preselected as an aptamer binding tobronopol from SEQ ID Nos. 189-301 flanked at its 5′-end with SEQ ID No.304 and at its 3′-end with SEQ ID No. 305) to cause dehybridization orotherwise spacing from the indicator to decrease quenching and increaseemitted signal due to excitation from the signal emitting label on theaptamer. FIG. 8 illustrates the fluorescence detected (shown at 560 nmfrom a 530 nm excitation source) at concentrations ranging from 0 to 200ppm. As illustrated, a concentration-dependent increase in fluorescencewas observed with increasing concentrations of the target bronopol.

Although the invention has been described with respect to specificembodiments thereof, these embodiments are merely illustrative, and notrestrictive of the invention. The description herein of illustratedembodiments of the invention, including the description in the Abstractand Summary, is not intended to be exhaustive or to limit the inventionto the precise forms disclosed herein (and in particular, the inclusionof any particular embodiment, feature or function within the Abstract orSummary is not intended to limit the scope of the invention to suchembodiment, feature or function). Rather, the description is intended todescribe illustrative embodiments, features and functions in order toprovide a person of ordinary skill in the art context to understand theinvention without limiting the invention to any particularly describedembodiment, feature or function, including any such embodiment featureor function described in the Abstract or Summary. While specificembodiments of, and examples for, the invention are described herein forillustrative purposes only, various equivalent modifications arepossible within the spirit and scope of the invention, as those skilledin the relevant art will recognize and appreciate. As indicated, thesemodifications may be made to the invention in light of the foregoingdescription of illustrated embodiments of the invention and are to beincluded within the spirit and scope of the invention. Thus, while theinvention has been described herein with reference to particularembodiments thereof, a latitude of modification, various changes andsubstitutions are intended in the foregoing disclosures, and it will beappreciated that in some instances some features of embodiments of theinvention will be employed without a corresponding use of other featureswithout departing from the scope and spirit of the invention as setforth. Therefore, many modifications may be made to adapt a particularsituation or material to the essential scope and spirit of theinvention.

Reference throughout this specification to “one embodiment”, “anembodiment”, or “a specific embodiment” or similar terminology meansthat a particular feature, structure, or characteristic described inconnection with the embodiment is included in at least one embodimentand may not necessarily be present in all embodiments. Thus, respectiveappearances of the phrases “in one embodiment”, “in an embodiment”, or“in a specific embodiment” or similar terminology in various placesthroughout this specification are not necessarily referring to the sameembodiment. Furthermore, the particular features, structures, orcharacteristics of any particular embodiment may be combined in anysuitable manner with one or more other embodiments. It is to beunderstood that other variations and modifications of the embodimentsdescribed and illustrated herein are possible in light of the teachingsherein and are to be considered as part of the spirit and scope of theinvention.

In the description herein, numerous specific details are provided, suchas examples of components and/or methods, to provide a thoroughunderstanding of embodiments of the invention. One skilled in therelevant art will recognize, however, that an embodiment may be able tobe practiced without one or more of the specific details, or with otherapparatus, systems, assemblies, methods, components, materials, parts,and/or the like. In other instances, well-known structures, components,systems, materials, or operations are not specifically shown ordescribed in detail to avoid obscuring aspects of embodiments of theinvention. While the invention may be illustrated by using a particularembodiment, this is not and does not limit the invention to anyparticular embodiment and a person of ordinary skill in the art willrecognize that additional embodiments are readily understandable and area part of this invention.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having,” or any other variation thereof, areintended to cover a non-exclusive inclusion. For example, a process,product, article, or apparatus that comprises a list of elements is notnecessarily limited only those elements but may include other elementsnot expressly listed or inherent to such process, process, article, orapparatus.

Furthermore, the term “or” as used herein is generally intended to mean“and/or” unless otherwise indicated. For example, a condition A or B issatisfied by any one of the following: A is true (or present) and B isfalse (or not present), A is false (or not present) and B is true (orpresent), and both A and B are true (or present). As used herein,including the claims that follow, a term preceded by “a” or “an” (and“the” when antecedent basis is “a” or “an”) includes both singular andplural of such term, unless clearly indicated within the claim otherwise(i.e., that the reference “a” or “an” clearly indicates only thesingular or only the plural). Also, as used in the description herein,the meaning of “in” includes “in” and “on” unless the context clearlydictates otherwise.

1. A method for utilizing a nucleic acid array comprising: providing asubstrate with a plurality of single-stranded nucleic acid ligandslocated and attached at predetermined addresses on said substrate;performing a hybridization of a single-stranded nucleic acid label toeach of said plurality of single-stranded nucleic acid ligands;incubating said substrate with a sample which may potentially contain atleast one target molecule which binds to at least one of said pluralityof single-stranded nucleic acid ligands; and performing a detectionoperation to detect potential changes in said hybridizations correlatedto said addresses; wherein said potential changes in said hybridizationsindicate binding of said at least one target molecule to at least one ofsaid plurality of single-stranded nucleic acid ligands.
 2. A method forselecting nucleic acid ligands comprising: providing a substrate with aplurality of single-stranded nucleic acid ligands located and attachedat predetermined addresses on said substrate, wherein each of saidpredetermined addresses comprises a different single-stranded nucleicacid ligand; performing a hybridization of a single-stranded nucleicacid label to each of said plurality of single-stranded nucleic acidligands; performing a binding and detection cycle comprising: incubatingsaid substrate with a sample which contains a target molecule which maybind to at least one of said plurality of single-stranded nucleic acidligands; and performing a detection operation to detect potentialchanges in said hybridizations at each of said addresses, wherein saidpotential changes in said hybridizations indicate binding of said targetmolecule to at least one of said plurality of single-stranded nucleicacid ligand; repeating said binding and detection cycle with a differenttarget molecule; and correlating which of said target molecules elicitedsaid potential changes in said hybridizations at each of said addressesto determine if any of said target molecules bind to any of saidplurality of single-stranded nucleic acid ligands.
 3. A method forutilizing a nucleic acid array comprising: providing a substrate with aplurality of single-stranded nucleic acid labels located and attached atpredetermined addresses on said substrate; performing a hybridization ofa single-stranded nucleic acid ligand to each of said plurality ofsingle-stranded nucleic acid labels, wherein the identity of saidsingle-stranded nucleic acid ligand hybridized at each predeterminedaddress is known; incubating said substrate with a sample which maypotentially contain at least one target molecule which binds to at leastone of said plurality of single-stranded nucleic acid ligands; andperforming a detection operation to detect potential changes in saidhybridizations correlated to said addresses; wherein said potentialchanges in said hybridizations indicate binding of said at least onetarget molecule to at least one of said plurality of single-strandednucleic acid ligands.
 4. The method of claim 1, wherein saidsingle-stranded nucleic acid ligands are selected for potentialconformational changes upon binding to at least one of said targetmolecules.
 5. The method of claim 1, wherein each of said hybridizationsof said single-stranded nucleic acid ligands and said single-strandednucleic acid labels comprise a signal interacting pair.
 6. The method ofclaim 1, wherein each of said hybridizations of said single-strandednucleic acid ligands and said single-stranded nucleic acid labelscomprise a radiation-emitting label:radiation-modulating proximityinteracting pair.
 7. The method of claim 1, wherein said potentialchanges are utilized to indicate presence of said target molecule in asample.
 8. The method of claim 1, wherein said potential changes areutilized to identify conformational changing aptamers from a collectionof aptamers.
 9. The method of claim 1, wherein said potential changesare utilized to identify aptamers which bind to said target moleculefrom a library of potential aptamers.
 10. The method of claim 1, whereinsaid potential changes are utilized to discriminate the relative bindingaffinity of potential aptamers to said target molecule.
 11. The methodof claim 1, wherein said potential changes are utilized to determine arelative abundance of said target molecule in a sample.
 12. The methodof claim 3, wherein said single-stranded nucleic acid labels include aspacing region between said substrate and a region that hybridizes tosaid single-stranded nucleic acid ligand.
 13. The method of claim 12,wherein said spacing region is between 5 and 20 nucleotides in length.14. The method of claim 2, wherein said single-stranded nucleic acidligands are selected for potential conformational changes upon bindingto at least one of said target molecules.
 15. The method of claim 2,wherein each of said hybridizations of said single-stranded nucleic acidligands and said single-stranded nucleic acid labels comprise a signalinteracting pair.
 16. The method of claim 2, wherein each of saidhybridizations of said single-stranded nucleic acid ligands and saidsingle-stranded nucleic acid labels comprise a radiation-emittinglabel:radiation-modulating proximity interacting pair.
 17. The method ofclaim 2, wherein said potential changes are utilized to indicatepresence of said target molecule in a sample.
 18. The method of claim 2,wherein said potential changes are utilized to identify conformationalchanging aptamers from a collection of aptamers.
 19. The method of claim2, wherein said potential changes are utilized to identify aptamerswhich bind to said target molecule from a library of potential aptamers.20. The method of claim 2, wherein said potential changes are utilizedto discriminate the relative binding affinity of potential aptamers tosaid target molecule.